Inhibition of pacap signalling for the prevention and treatment of thrombocytopenia

ABSTRACT

The present invention discloses the use of inhibitors and/or antagonists of PACAP signalling for the manufacture of a medicament for the prevention or treatment of decreased blood platelet numbers (thrombocytopenia).

The present application is a continuation of application Ser. No.10/542,238, filed Jul. 15, 2005 (allowed), which is a U.S. nationalphase of PCT/EP2004/001209, filed Jan. 16, 2004, which claims benefit ofGB application Nos. 0300934.7, 0307667.6, and 0310037.7, filed Jan. 16,2003, Apr. 3, 2003 and Apr. 3, 2003, respectively.

FIELD OF THE INVENTION

This invention relates to compounds useful in the control of the primaryhaemostasis and the modulation of platelet number, as well as methods ofuse of these compounds.

BACKGROUND OF THE INVENTION

The initial response to interruption of continuity of a blood vessel isdefined as primary haemostasis. Platelets play a major role in thepathophysiology of primary haemostasis. The clinical importance ofplatelets first became obvious when thrombocytopenic patients, who lateron were diagnosed as having immune mediated thrombocytopenia (ITP), hadthrombocytopenic purpura. Platelets participate in haemostasis bysealing vascular injuries and by fostering the process of bloodcoagulation. Not only the number of the platelets is important(thrombocytopenia for whatever reason) but also their intrinsic functionupon activation: change in platelet shape, adhesion, aggregation andsecretion are prerequisites for normal haemostasis. Congenital oracquired disorders interfering with one of the platelet functions canlead to mild to even severe bleeding problems.

Prevention and treatment of bleeding in patients with thrombocytopeniaor thrombocytopathia is therefore based on platelet transfusion ormedication interfering with platelet number and/or function.

Platelets also play a role in the development of arterial thrombosis.Disruption of the endothelial cell lining of the vessels exposesadhesive proteins within the subendothelial matrix, leading to plateletattachment. Thereafter, platelet spreading, as well as plateletsecretion occurs. The secretion of the content of platelet granules canstimulate circulating platelets to acquire new adhesive properties.Finally, stimulated platelets interact with each other during plateletaggregation and a platelet-rich thrombus is formed, which can compromisethe patency of blood vessels. Furthermore activated platelets acceleratethe rate of activation of coagulation proteins. Phospholipids on theplatelet surface facilitate thrombin generation and fibrin strandformation.

Arterial and venous thrombosis and their complications includingischemic stroke, acute myocardial infarction and venous thromboembolism,represent the major cause of morbidity and mortality in developedcountries.

Prevention and treatment of thrombosis are therefore based onadministration of antiplatelet drugs, anticoagulants or thrombolytictherapy or combinations thereof.

The pituitary Adenylyl Cyclase Activating Peptide (PACAP 1-38) is a38-amino acid peptide that was first isolated from ovine hypothalamicextracts on the basis of its ability to stimulate cAMP formation inanterior pituitary cells [Miyata A. et al. (1989) Biochem Biophys ResCommun 164, 567-574; Vaudry D. et al. (2000) Pharm Rev 52, 269-324].PACAP is a member of the Vasoactive Intestinal Polypeptide(VIP)-glucagon-growth hormone releasing factor-secretin superfamily. Itsrole in biology is probably crucial, since the sequence of PACAP ishighly conserved during the evolution from protochordate to mammals.PACAP is widely expressed and occurs in the central and peripheralnervous system, the urogenital system, the gastro-intestinal tract, andin several endocrine glands. PACAP receptors are also widely distributed(Vaudray et al. cited supra). Two classes of PACAP binding sites havebeen characterized based on their relative affinities for PACAP and VIP:type I binding sites with high affinity for PACAP (Kd=0.5 nM) and muchlower affinity for VIP (Kd>500 nM) and type II binding sites, which arewidely distributed in various peripheral organs, characterised bysimilar affinities for PACAP and VIP (Kd=1 nM). Molecular cloning ofPACAP receptors has demonstrated the existence of three distinctreceptor subtypes that are abundantly spread in many tissues: thePACAP-specific PACAP receptor, coupled to different signal transductionsystems, and two PACAP: VIP-indifferent receptors (VPAC1 and VPAC2, alsoreferred to as VIPR1 and VIPR2, respectively), which are primarilycoupled to adenylyl cyclase.

The exact biological and pharmacological function of PACAP is presentlybeing investigated in many organs and tissues as in endocrine glands,central nervous system, respiratory system, cardiovascular system andgastrointestinal tract. Although extensive studies have also beenperformed on its function in the immune system, limited data areavailable concerning its function on haemostasis. An effect of PACAP onaggregation and metabolism of isolated platelets has been described [Kiset al. (1999) Prostaglandins Other Lipid Mediat. 58:103-112 and Ichikiet al. (1992) Biochem Biophys Res Commun. 187, 1587-1593]. Theoccurrence of the VPAC1 receptor on platelets which binds both VIP asPACAP has been reported (Park et al. (1996) Blood 87, 4629-4635).

SUMMARY OF THE INVENTION

The invention relates to the use of an inhibitor of PACAP signalling forthe manufacture of a medicament for the prevention or treatment ofthrombocytopenia. Herein the inhibitor of PACAP signalling can targetexpressed PACAP, expressed VIP or a receptor for PACAP. A receptor forPACAP can be the PACAP receptor (PACAPR), VPAC1 or VPAC2. The inhibitorcan be an antagonist which interferes with PACAP, VIP or a PACAPreceptor protein. Alternatively the inhibitor can inhibit thetranscription or translation of PACAP, VIP or a receptor for PACAP.Examples of inhibitors of transcription and translation include anantisense molecule, an RNAi, an aptamer, a small molecule, an antibody,a ribozyme, a transdominant receptor, and a tetrameric peptide. Specificcompounds having an inhibitory effect on PACAP signalling includemax.d.4 5, PACAP6-38, [4CI-D-Phe6, Leu17]VIP, VIP(10-28), cyclic lactamanalogs of PACAP, [AcHis(1), D-Phe(2), Lys(15),Leu(17)]VIP(3-7)/GRF(8-27), PACAP receptor blocking Cyclic lactam PACAPanalogs, N-terminal truncated or substituted VIP peptide PACAP receptorblockers, neutralising antibodies against VPAC(1), and neutralisingaptamer against VPAC(1) receptor, [4CI-D-Phe6, Leu17]VIP, VIP(10-28).

The invention also relates to a pharmaceutical composition comprising aninhibitor of PACAP signalling and an additional compound for enhancingmegakaryocyte maturation such as thrombopoetin or Interleukin 11.

The invention also relates to the use of pituitary adenylyl cyclaseactivating peptide (PACAP), its derivatives, mimetics, antibodies orinhibitors for modulating the primary haemostasis or thrombocytopoiesisor the use of agonists or antagonists of the platelet receptor, VPAC1,to modulate the primary haemostasis or thrombocytopoiesis.

The present invention further relates to a method for prevention as aprophylactic and for the treatment of either thrombosis or bleedingbased on administration of pituitary adenylyl cyclase activating peptide(PACAP) mimetics or inhibitors respectively.

The present invention shows that human platelets express the VPAC1receptor. The present invention also shows that PACAP has an importantfunction in primary haemostasis: platelet number as well as plateletfunction are highly influenced by PACAP.

The invention provides compositions and methods useful for activatingthrombocytopoiesis in mammals, including humans. The invention appliesto human and veterinary applications. The inventive composition andmethod have been shown to be especially effective in treating platelethypofunction.

In addition, the present invention finds utility in e.g., radiationtreatment or chemotherapeutic treatment or sickness caused by radiationaccidents, where prevention or treatment of thrombocytopenia, anemia andneutropenia is a significant concern. The depletion of hematopoieticprecursors in the bone marrow associated with chemotherapy andirradiation results in hemorrhagic and infectious complications. Severesuppression of the hematopoietic system is a major factor in limitingchemotherapy use and dose escalation.

Compounds useful for exercising the thrombocytopoietic treatment or themanufacturing of a medicament for platelet hypofunction of presentinvention are thus compounds that inhibit the activity of PACAP or VIPand more specifically that antagonise the platelet receptor for PACAP.

DETAILED DESCRIPTION Brief Description of the Figures

FIG. 1. Family presentation. Squares, male; circles, female; filledsymbols, affected individuals; open symbols, unaffected individuals.Patient (VI:1) is the propositus. Black filled symbols represent memberswith severe mental retardation and a partial trisomy 18p and monosomy20p, striped boxes represent members with borderline IQ and the balancedtranslocation t(18;20) (p21;p13) while question marks stand for memberswith unexplained mental retardation but unknown karyotype.

FIG. 2. Platelet aggregation and adenylyl cyclase activity. Measurementsof cAMP levels under basal conditions (2A) performed in duplicate (leftpanel) or after stimulation with Iloprost (1 ng/ml) (2B) for varioustime intervals (right panel) in platelets from VI:1 (v) or an unrelatedcontrol (CrI)(X). Measurements of cAMP levels under basal conditionsperformed in duplicate (left panel) or after stimulation withisoproterenol (1 μM) for various time intervals (right panel) infibroblasts from VI:1, a patient with trisomy 18 (σ) or an unrelatedcontrol (CrI) (X). All cAMP measurements were performed in the presenceof the phosphodiesterase inhibitor IBMX (400 μM).

FIG. 3. PACAP detection in fibroblasts and plasma. 3A: Semi-quantitativeRT-PCR using 20 cycles and 17 showed PACAP(1-38) overexpression infibroblasts from patient VI:1 compared with two controls. β-actin is theinternal control. 3B: PACAP mRNA detection in fibroblasts,megakaryocytic cell lines DAMI, MEG-01 and K562, and platelets wasperformed by RT-PCR. 3C: Immunoblot analysis of the VPAC1 receptor (58kDa) in platelets from two unrelated controls and patient VI:1. 3D PACAPdetections by ELISA in plasma from citrate (left panel) or ACD (acidcitrate dextrose) (right panel) blood show pronounced or moderatelyincreased PACAP level in respectively VI:1 (v) and V:4 (σ) or IV:5 (λ)and V:3 (ν) versus a citrated plasma pool (T) or IV:6 (Γ).3E. PACAPlevels detected by ELISA in plasma (I) or serum (n) from mice (n=3). 3F.Collagen induced aggregation of control platelets in plasma from acontrol or from patient IV:1 (two experiments shown).

FIG. 4. Role of PACAP(6-38) in platelet aggregation. 4A: Left panel: themean bleeding time (sec)+/−SD for 10 animals in each group (unpairedT-test; p=0.0001) from either wild type (WT) versus PACAP overexpressing(PACAP-Tg) mice. 4A Right panel: the collagen-induced (5 mg/ml) plateletaggregation in hirudinized PRP from 2 WT and two PACAP-Tg mice areshown. 4B: The left panel shows the dose-dependent stimulation byPACAP(6-38) of the collagen-induced (0.2 μg/ml) aggregation of humanplatelets; representative tracings of five separate experiments. Theeffect of PACAP (6-38) on the collagen-induced platelet aggregation (2microgramg/ml) for patient VI:1 is illustrated in the right panel. 4C:The platelet aggregation inhibition test with collagen (2 μg/ml) anddifferent concentrations of Iloprost (ng/ml) as indicated in the absence(left panels) and presence (right panels) of PACAP(6-38) for a control(upper panels) or patient VI:1 (lower panels).

FIG. 5. Effect of anti-PACAP antibodies in mice. Platelet aggregationwas performed in PRP pooled from five mice of each group with 250×10³plt/μl. 5A: The platelet aggregation inhibition test with collagen (2μg/ml) and preincubation of Iloprost (10 ng/ml) for mice injected withthe indicated antibodies. 5B: Platelet aggregation induced with a lowconcentration of collagen (0.2 μg/ml) for mice injected with theindicated antibodies. 5C: Stimulatory effect of a polyclonal anti-PACAPantibody (10 μg/ml) on collagen-induced (0.35 μg/ml) plateletaggregation.

Effect of PACAP on thrombopoiesis. 5D: FACS analysis showed a reducedexpression of glycoprotein IX (CD42a) in platelets from patient VI:1versus control platelets (left panel) and a reduced expression ofglycoprotein IIIA (CD61) in the megakaryocytes of the PACAPoverexpressing mice compared to cells of the control mice (right panel).These experiments were repeated twice with identical results. 5E:Electron micrograph of megakaryocyte progenitor cell. Specific granulesare seen as well as a dense core vesicle (arrow). A few endoplasmicreticulum cisternae are obvious. Original magnification: ×18.500. 5F:Mean platelet number+/−SD in mice (n=5) injected with either polyclonalanti-PACAP (group A) or monoclonal anti-human vWF (75H₄B12) (group B)antibody, determined on the indicated days.

FIG. 6. The mean platelet number per μl for mice (n=5) injected witheither polyclonal anti-PACAP (6A) or an irrelevant anti-β2-glycoproteinI (6B) antibody was determined 14 days after the first antibodyinjection.

FIG. 7. Mean platelet number+/−SD for mice (n=5) injected with eitherpolyclonal anti-PACAP (bars indicated with A) or anti-vWF (75H₄B12)(bars indicated with B) antibody determined at the indicated days.

FIG. 8. Mean platelet number/μl+/−SD for mice (n=5) injected with eitherpolyclonal anti-PACAP (bars indicated with A) or an irrelevant anti-XLαs(bars indicated with B) polyclonal antibody determined at the indicateddays. Data represent separated experiments A (top figure) and B (bottomfigure).

DEFINITIONS

“PACAP” refers to Pituitary Adenylate Cyclase-Activating Polypeptide andrefers to the mature and processed versions of the mature PACAP asPACAP(1-48) and more particularly to processed isoforms PACAP(1-38)HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK-NH2 [SEQ ID NO:1] and PACAP(1-27)HSDGIFTDSYSRYRKQMAVKKYLAAVL-NH2 [SEQ ID NO:2]. PACAP refers to PACAP ofvertebrates, including mammalian PACAP and human PACAP.

“VIP” refers to Vasoactive Intestinal Peptide (VIP), a 28-amino acidpeptide which exhibits a wide variety of biologic actions. Because VIPshows similarities to glucagon, secretin and gastric inhibitory peptide(GIP), it has been considered a member of the glucagon-secretin family.The primary translation product of the mRNA encoding VIP (prepro-VIP)has a molecular weight of 20 daltons.

“receptor for PACAP” refers to a receptor which is bound and activatedby PACAP including the PACAP receptor (PACAPR) [OMIM 102981] which isspecific for PACAP but also to VPAC1 (VIPR1) [OMIM 192321] and VPAC2(VIPR2) [OMIM 601970] receptors which bind both PACAP and VIP(Vasoactive Intestinal peptide).

As used herein, “thrombocytopenia” is any disorder in which the plateletlevel in the affected individual fall below a normal range of plateletnumbers for that individual, due to disturbance in productiondistribution or destruction. In humans, normal blood platelet levelsrange from about 150.000 to 300.000 per microliter peripheral blood.With a platelet level of 100.000 per microliter patients have noabnormal bleeding even with major surgery; with a platelet count of50.000 to 100.000 per microliter, patients may bleed longer than normalwith severe trauma; with a platelet count of 20.000 to 50.000 permicroliter, bleeding occurs with minor trauma but spontaneous bleedingis unusual; with a platelet count of less than 20.000, patients may havespontaneous bleeding and when the platelet count is less than 10.000 permicroliter, patients are at high risk for severe bleeding.

Thrombocytopenia also refers to a decrease in platelet number in anindividual when compared to the platelet number measured at a certainreference point in that individual. The decrease in platelet number inthe individual can be a decrease in more than 20%, 30%, 40%, 60%, 80%,90%, 95% or even more, compared to value at the reference point. Adecrease in platelet number when compared to the platelet numbermeasured at a certain reference point, can in certain individuals beaccompanied with changes in bleeding, while in other individuals acomparable decrease will not be accompanied with changes in bleeding.The reference point mentioned, can be for instance the start of atherapy such as a radiation or chemotherapy.

Thrombocytopenia includes infection-induced thrombocytopenia,treatment-induced thrombocytopenia and others.

“Infection-induced thrombocytopenia” is a disorder characterised by alowered level of platelets in peripheral blood, which is caused by aninfectious agent such as a bacteria or virus.

“Treatment-induced thrombocytopenia” is a disorder characterised by alowered level of platelets in peripheral blood which is caused bytherapeutic treatments such as gamma irradiation, therapeutic exposureto radiation, cytotoxic drugs, chemicals containing benzene oranthracene and even some commonly used drugs such as chloramphenicol,thiouracil, and barbiturate hypnotics.

“Other types of thrombocytopenia” comprise disorders characterised by alow level of platelets in peripheral blood, which are caused by anymechanism other than infectious agents or therapeutic treatments causingthrombocytopenia. Factors causing this type of thrombocytopenia include,but are not limited to, rare bone marrow disorders such as congenitalamegakaryocytic hypoplasia and thrombocytopenia with absent radii (TARsyndrome), an increase in spleen size, or splenomegaly, caused by portalhypertension, secondary to liver disease, or macrophage storagedisorders such as Gauchers disease, autoimmune disorders such asidiopathic or immune thrombocytopenic purpura (ITP), vasculitis,hemolytic uremic syndrome, thrombotic thrombocytopenic purpura (TTP),disseminated intravascular coagulation (DIC) and prosthetic cardiacvalves. ITP is by far the most frequent type in this group ofthrombocytopenias.

A “subject having thrombocytopenia” is a subject having any type ofthrombocytopenia and includes but is not limited tonon-chemotherapeutic-induced thrombocytopenia, orchemotherapeutic-induced thrombocytopenia.

“A subject at risk of developing thrombocytopenia” is a subject who hasa high probability of acquiring or developing thrombocytopenia. Forexample, a patient with a malignant tumour who is prescribed achemotherapeutic treatment is at risk of developing treatment-inducedthrombocytopenia and a subject who has an increased risk of exposure toinfectious agents is at risk of developing infection-inducedthrombocytopenia.

“PACAP signalling” refers to the binding and activation of a receptorfor PACAP. It thus relates to the binding of PACAP to the PACACreceptor, VPAC1 or VPAC2 r and subsequent activation. Moreover PACAPsignalling also includes binding and activation of VPAC1 and VPAC2 byVIP. In a particular embodiment “PACAP signalling” relates to signallingvia the VPAC1 receptor present on megakaryocytes in bone marrow.

“Inhibition of PACAP signalling” refers to the inhibition of the bindingof PACAP or VIP to a receptor for PACAP, which includes inhibition ofthe production and/or activity of the ligands PACAP and/or VIP (PACAP orVIP inhibition by PACAP and/or VIP inhibitors) and inhibition of theproduction or function of one or more receptors for PACAP or the bindingof PACAP or VIP thereto (PACAP receptor inhibition), e.g. by antibodies,antagonists, soluble receptors, antisense etc, as detailed herein. Thesemolecules are also generally referred to herein as ‘inhibitors of PACAPsignalling’.

The term ‘anti-PACAP antibody or antibodies’ relates to an antibody orantibodies characterised as being specifically directed against PACAP,VIP or any functional derivative thereof, with said antibodies beingpreferably monoclonal antibodies or an antigen-binding fragment thereof,of the F(ab′)₂, F(ab) or single chain Fv type, or any type ofrecombinant antibody or antibody fragment derived thereof. Preferably,said antibody or fragment thereof is humanized or is in other waysappropriate for injection into the subject to be treated therewith.

The term ‘anti-PACAP Receptor antibody or antibodies’ relates to anantibody or antibodies characterised as being specifically directedagainst one or more of thereceptors for PACAP (PACAPR, VPAC1 and VPAC2)with said antibodies being preferably monoclonal antibodies; or anantigen-binding fragment thereof, of the F(ab′)₂, F(ab) or single chainFv type, or any type of recombinant antibody derived thereof.

These antibodies of the invention, including specific polyclonalantisera prepared against a receptor for PACAP, the ligands PACAP, VIP,or any functional derivative thereof, have no cross-reactivity to otherproteins. The monoclonal antibodies of the invention can, for instance,be produced by any hybridoma produced according to classical methodsusing splenic cells of an animal, particularly of a mouse or ratimmunized against a receptor for PACAP, PACAP, VIP, a molecule involvedin PACAP signalling, or any functional derivative thereof, and cells ofa myeloma cell line, and can be selected by the ability of the hybridomato produce the monoclonal antibodies recognizing a receptor for PACAP,PACAP, VIP, the molecule involved in PACAP signalling, or any functionalderivative thereof which has been initially used for the immunization ofthe animals.

The monoclonal anti-PACAP or antibodies against a receptor for PACAPaccording to the invention may be humanized versions of the non-humanmonoclonal antibodies, made by means of recombinant DNA technology,departing from the mouse and/or human genomic DNA sequences coding for Hand L chains or from cDNA clones coding for H and L chains.Alternatively the monoclonal antibodies according to this embodiment ofthe invention may be human monoclonal antibodies. Such human monoclonalantibodies are prepared, for instance, by means of human peripheralblood lymphocytes (PBL) repopulation of severe combined immunedeficiency (SCID) mice as described in PCT/EP99/03605 or by usingtransgenic non-human animals capable of producing human antibodies asdescribed in U.S. Pat. No. 5,545,806. Also fragments derived from thesemonoclonal antibodies such as Fab, F(ab)′2 and ssFv (“single chainvariable fragment”), provided they have retained the original bindingproperties, form part of the present invention. Such fragments arecommonly generated by, for instance, enzymatic digestion of theantibodies with papain, pepsin, or other proteases. It is well known tothe person skilled in the art that monoclonal antibodies, or fragmentsthereof, can be modified for various uses. An appropriate label of theenzymatic, fluorescent, or radioactive type can label the antibodiesinvolved in the invention.

“stimulation of PACAP signalling” refers to the stimulation of a signalcaused by the binding of PACAP, VIP or an agonist thereof to a receptorfor PACAP which includes stimulation of the production or increase ofthe presence of the ligands PACAP and/or VIP, or agonists thereof orstimulation of the production or activity of one or more of a receptorfor PACAP or the binding of PACAP or VIP thereto (PACAP receptorstimulation), e.g. by agonists, co-expression of PACAP or VIP-likemolecules, etc, as detailed herein. These molecules are also generallyreferred to herein as ‘stimulators of PACAP signalling’.

The term “pharmaceutically acceptable” is used to indicate that acompound used in the manufacture of the medicament is appropriate, forexample with respect to specific activity or purity. The term“treatment” or “prevention” refers to any process, action, application,therapy, or the like, wherein a subject or individual is subjected tomedical aid with the object of improving or maintaining the mammal'scondition, directly or indirectly. Subject, individual or patient refersto a mammal and includes a laboratory animal, pet, agriculture animal;examples include dogs, cats, horses, rodents (rats, mice, hamsters,guinea pigs), cattle, pigs, rabbits, goats, among others. A mammal alsorefers to monkeys and primates, including humans.

The present invention relates to the modulation of platelet function andnumber through the inhibition of PACAP signalling.

In one aspect, the present invention relates to the inhibition of PACAPsignalling by targeting PACAP and/or VIP in a subject and to compoundsuseful therefore.

In a particular embodiment it relates to PACAP and/or VIP inhibition inthe peripheral blood of a subject.

Compounds which target expressed PACAP and/or VIP (referred to herein asexamples of PACAP and/or VIP inhibitors) include polyclonal ormonoclonal antibodies or antibody fragments against PACAP or VIP. Othercompounds targeting expressed PACAP and/or VIP include soluble fragmentsof PACAP receptors. Thus more specifically the invention also relates tomolecules that neutralize the activity of PACAP and/or VIP byinterfering with its dimerisation, receptor-binding and/orreceptor-binding-mediated signal transduction. By molecules it is meantpeptides, tetrameric peptides, proteins, organic molecules, mutants ofPACAP or VIP, soluble receptors of the PACAP receptor (PACAPR), VPAC1 orVPAC2 and any fragment or homologue thereof having the same neutralizingeffect as stated above.

In another aspect, the present invention relates to the inhibition ofPACAP signalling by targeting one or more of a receptor for PACAP(capable of binding PACAP) and to compounds useful therefore.

For instance, certain (poly)peptides are known to be PACAP receptor(PACAPR) antagonists such as max.d.4 5 (Sakashita et al. Br. (2001) J.Pharmacol, 132: 1769-1776) and PACAP6-38 (Tohei et al. (2001)Neuro-endocrinol. 73, 68-74). Compounds with type II PACAP receptor(VPAC1 and VPAC2) antagonist properties comprise several N-terminaltruncated or substituted VIP peptides such as [4CI-D-Phe6, Leu17]VIP,VIP(10-28) (Pandol et al. (1986) Am. J. Physiol. 250: G553 G557; Turneret al. (1986), Peptides 7: 849-854; Gozes et al. (1995) Cell MolNeurobiol. 15, 675-687; Gourlet et al. (1997) Peptides 18, 1555-60),cyclic lactam analogs of PACAP (Bitar at al. (1994) Peptides 15:461-466). VPAC1 receptor selective antagonists known are for instance[AcHis(1), D-Phe(2), Lys(15), Leu(17)]VIP(3-7)/GRF(8-27) (Lema-Kisoka Ret al. (2001) cited supra). Other PACAP receptor agonists areneutralising antibodies against VPAC1 or aptamers (3-dimensional nucleicacids that bind to molecular targets in a manner similar to antibodies)that bind to VPAC1 receptor (or against PACAP) thereby neutralising itsactivity. Antagonising peptides MAX65 and MAX65 NH₂ are described inU.S. Pat. No. 6,017,533. VIP antagonising peptides with modified aminoand carboxyterminal groups are described in WO95/21294. Other VIPantagonising compounds are described in U.S. Pat. No. 5,217,953, U.S.Pat. No. 5,565,242 and U.S. Pat. No. 6,630,124.

Small molecules that act as a PACAP receptor antagonist, e.g. smallorganic molecules and other drug candidates can be obtained, forexample, from combinatorial and natural product libraries. Randompeptide libraries, such as the use of tetrameric peptide libraries suchas described in WO0185796, consisting of all possible combinations ofamino acids attached to a solid phase support may be used in identifyingcompounds useful in the present invention. Also transdominant-negativemutant forms of PACAP-receptors (e.g. a transdominant-negative receptorof VPAC1 or VPAC2 can be used to inhibit the signal transduction ofPACAP or VIP.

Thus, one aspect of inhibition of PACAP signalling refers to inhibitionat the protein level (PACAP or VIP or any of a receptor for PACAP). Theinhibition of PACAP signalling leads to a diminished interaction withits receptor and an inhibition of signal transduction. Preferably saidinhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or evenhigher. The inhibition of compounds on PACAP signalling can be measuredfor example by platelet aggregation or measuring the release ofcompounds from platelets such as ATP or serotonin. The biologicalactivity of other potential PACAP agonists or antagonist can bedetermined by (1) determining whether the compound binds to receptorsfor PACAP (Gottschall, et al. (1990) Endocrinology 127, 272; EPApplication 529 487) and (2) determining whether the compound stimulatesthe production or release of PACAP. This can be assayed on isolatedplatelets or megakaryocytes or can be assayed in a cell culture systemwherein cells are transfected with PACAPR, VPAC1 or VPAC2.

In another aspect, the present invention relates to the inhibition ofPACAP signalling by preventing expression (transcription and/ortranslation) of the PACAP and/or VIP gene and/or by preventingexpression of a receptor for PACAP in blood platelet precursors and tocompounds useful therefore. This inhibition is particularly effectivewhen done in hematopoietic stem cells or in megakaryocytes or precursorcells thereof.

Examples of molecules that are useful for this aspect of the inventionare anti-sense RNA and DNA molecules (e.g. polynucleotide sequences) orribozymes that function to inhibit the translation.

Small molecules can also interfere by binding on the promoter region ofa gene and inhibiting the binding of a transcription factor on saidpromoter region so that no mRNA is produced.

Also within the scope of the invention is the use of oligoribonucleotidesequences that include anti-sense RNA and DNA molecules and ribozymesthat function to inhibit the translation of VPAC1 or VPAC2 mRNA or PACAPmRNA or VIP mRNA. Anti-sense RNA and DNA molecules act to directly blockthe translation of mRNA by binding to targeted mRNA and preventingprotein translation. With regard to antisense DNA,oligodeoxyribonucleotides derived from the translation initiation sitecan be used. Ribozymes are enzymatic RNA molecules capable of catalysingthe specific cleavage of RNA. The mechanism of ribozyme action involvessequence specific hybridisation of the ribozyme molecule tocomplementary target RNA, followed by an endonucleolytic cleavage.Within the scope of the invention are engineered hammerhead motifribozyme molecules that specifically and efficiently catalyseendonucleolytic cleavage of VPAC(1) RNA, PACAP RNA or VIP RNA sequences.Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for predicted structuralfeatures such as secondary structure that may render the oligonucleotidesequence unsuitable. Both anti-sense RNA and DNA molecules and ribozymesof the invention may be prepared by any method known in the art for thesynthesis of RNA molecules. These include techniques for chemicallysynthesizing oligodeoxyribonucleotides well known in the art such as forexample solid phase phosphoramidite chemical synthesis. Alternatively,RNA molecules may be generated by in vitro and in vivo transcription ofDNA sequences encoding the antisense RNA molecule. Such DNA sequencesmay be incorporated into a wide variety of vectors, which incorporatesuitable RNA polymerase promoters such as the T7 or SP6 polymerasepromoters. Alternatively, antisense cDNA constructs that synthesizeanti-sense RNA constitutively or inducible, depending on the promoterused, can be introduced stably into cell lines.

Inhibition of PACAP signalling through gene transcription and/ortranslation by these compounds according to the present invention is atleast 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even higher and can bemeasured by lowered mRNA or protein levels.

The invention in one aspect relates to a method for increasing ormaintaining platelet counts in a subject having thrombocytopenia or asubject at risk of developing thrombocytopenia, by administering to thesubject an inhibitor of PACAP signalling.

Thus the invention relates to the use of an inhibitor of PACAPsignalling for the manufacture of a medicament for the prevention ortreatment of thrombocytopenia.

The prevention or treatment against thrombocytopenia can be aimed at anincreased level of active platelets. This means that platelet numberbecomes higher after administration of the inhibitor than prior to theadministration. An amount effective to increase platelet counts in thesubject is an amount, which causes an increase in the amount ofcirculating platelet levels. The actual levels of platelets achievedwill vary depending on many variables such as the initial status of theimmune system in the subject, i.e., whether the subject has mild tosevere thrombocytopenia (e.g., resulting from an autoimmune disease orsplenic sequestration). In general, the platelet levels of a subject whohas severe thrombocytopenia will initially be very low. Any increase inthe platelet levels of such a subject, even an increase to a level thatis still below a normal level, can be advantageous to the subject, sinceat the same time platelet function will be upregulated. Theadministration of the inhibitor aims to increase the number of plateletsby at least 20, 50, 75 or 100%. Depending on the initial status of theindividual (severe or low thrombocytopenia or normal platelet number),the individual will, after the administration of the inhibitor, displaya low thrombocytopenia, a normal platelet number or a platelet number ofnormal levels). Alternatively, and more particularly in case ofprevention of thrombocytopenia e.g. in combination with or beforechemotherapy, administration of an inhibitor of the PACAP signallingpathway can be aimed at a maintenance of the number of active platelets(i.e. preventing a significant decrease in the number of plateletsexpected as a result of chemotherapy).

The compositions for inducing platelet production, comprising aneffective quantity of a PACAP inhibitor and/or VIP inhibitor and/or aPACAP receptor inhibitor can be in admixture with pharmaceuticallyacceptable diluents, carriers or excipients. This property ofstimulating platelet production of the molecule should render it auseful adjunct in the therapy of patients suffering from acutethrombocytopenia, for example, as a result of chemo- or radiotherapy ofvarious cancers. Currently, such patients are at grave risk whencirculating platelet levels are depressed to levels wherebythrombogenesis is precluded. In a particular embodiment the presentinvention relates to a pharmaceutical composition comprising aninhibitor of PACAP signalling and an additional compound for enhancingmegakaryocyte maturation such as thrombopoetin or Interleukin 11.

The term medicament relates to a composition comprising molecules asdescribed above and a pharmaceutically acceptable carrier or excipient(both terms can be used interchangeably) to treat diseases as indicatedabove. Suitable carriers or excipients known to the skilled man aresaline, Ringer's solution, dextrose solution, Hank's solution, fixedoils, ethyl oleate, 5% dextrose in saline, substances that enhanceisotonicity and chemical stability, buffers and preservatives. Othersuitable carriers include any carrier that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids and amino acid copolymers. The‘medicament’ may be administered by any suitable method within theknowledge of the skilled person. The preferred route of administrationis parenterally. For parenteral administration, the medicament of thisinvention will be formulated in a unit dosage injectable form such as asolution, suspension or emulsion, in association with thepharmaceutically acceptable excipients as defined above. However, thedosage and mode of administration will depend on the individual.Generally, the medicament is administered so that the compound, protein,polypeptide, peptide of the present invention is given at a dose between1 μg/kg and 10 mg/kg, more preferably between 10 μg/kg and 5 mg/kg, mostpreferably between 0.1 and 2 mg/kg. Preferably, it is given as a bolusdose. Continuous infusion may also be used and includes continuoussubcutaneous delivery via an osmotic minipump. If so, the medicament maybe infused at a dose between 5 and 20 μg/kg/minute, more preferablybetween 7 and 15 μg/kg/minute.

Another aspect of administration for treatment is the use of genetherapy to deliver the above mentioned anti-sense gene or functionalparts of the VPAC(1) gene, PACAP gene or VIP gene or a ribozyme directedagainst the VPAC(1) mRNA, PACAP mRNA, VIP mRNA or a functional partthereof or a genetic construct encoding a transdominant-negative mutantform of VPAC(1)-receptors. Gene therapy means the treatment by thedelivery of therapeutic nucleic acids to patient's cells (extensivelyreviewed in Lever and Goodfellow (1995) Br. Med. Bull. 51, 1-242; Culveret al. (1995) Br. Med. Bull. 51, 192-204; Ledley, F. D. (1995) Hum. GeneTher. 6, 1129). Gene therapy requires a method for delivering genes tothe patient's cells and additional methods to ensure the effectiveproduction of any therapeutic genes. There are two general approaches toachieve gene delivery; these are non-viral delivery and virus-mediatedgene delivery.

Another aspect of the invention relates the use of an inhibitor of PACAPsignalling, i.e. a compound that inhibits the expression and/or activityof PACAP or VIP gene or a PACAP or VIP protein, for the manufacture of amedicament for treatment of platelet hypofunction. This is based on thefinding that platelet hypofunction can be treated (activated) byinhibiting the expression and/or activity of PACAP.

An embodiment of the present invention relates to the use of moleculeswhich comprise a region that can specifically bind to PACAP or to one ofits receptors such as (VPAC(1) receptor) and which consequentlyinterfere with the binding of PACAP and/or VIP to the VPAC(1) receptor,interfering with the signal transduction of PACAP and/or VIP. Saidmolecules can be used for the manufacture of a medicament for treatmentof platelet hypofunction.

Yet another aspect of the present invention relates to the use ofstimulators of PACAP signalling in the prevention and treatment ofthrombolytic disorders. Examples of stimulators of PACAP signallinginclude the 38-amino acid form of PACAP (PACAP38),HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK-NH2 and PACAP27, a 27-residuealpha-amidated polypeptide, HSDGIFTDSYSRYRKQMAVKKYLAAVL-NH2, which areboth potent agonists of PACAPNIP receptors. Based on their bindingaffinity and adenylyl cyclase stimulating properties, PACAP 2-38, PACAP2-27, AcHIS1 PACAP1-27, Ala2 PACAP(1-27) have also been demonstrated tobe potent agonists of PACAP/VIP receptors (Vaudry D. et al. citedsupra).

Amino acid substitutions and additions of a fatty acyl moiety have ledto the development of lipophilic VIP derivatives that exhibit enhancedpotency and specificity for VPAC receptors (Gozes and Fridkin, (1992) J.Clin. Invest 90: 810-814; Gozes et al. (1995) Cell Mol. Neurobiol. 15:675-687; Gourlet et al. (1998) Eur. J. Pharmacol 354: 105-111).

PACAP receptor (PACAPR) selective agonists are, for instance, thecompounds of the group consisting of the PACAP, VIP, [K(15), R(16),L(27)]VIP(1-7)/GRF(8-27), [R(16)]ChSn), (Lundberg et al. (2001)Endocrinology 142, 339-347; Lema-Kisoka R et al. (2001) Peptides 22,2155-2162), [(11,22,28)Ala]-VIP (Anderson C. M. et al. (2003) Br. J.Pharmacol. 138, 564-73).

Agonists of the type II PACAP receptor, VPAC1 are for instance compoundsof the group consisting of [R(16)]-PACAP(1-23) and [(L22)]-VIP (VanRampelbergh et al. (2000) Br. J. Pharmacol. 130: 819-26) and (Lys15,Arg16, Leu27)VIP1-7 GRF8-27 (Moody T W et al. (2000) Ann-N.Y. Acad. Sci.921, 26-32).

An important embodiment of this aspect of the present invention relatesto compositions comprising an activator of PACAP and/or a PACAP receptoragonist, preferably a VPAC(1) receptor agonist for treatment(particularly the prevention or suppression) of platelet hyperfunctionin a subject. It can be a pharmaceutical composition, which comprises anamount of PACAP or a mimetic thereof effective for blocking orpreventing thrombosis in a subject, and a pharmaceutically effectivecarrier. This pharmaceutical composition can be used to treat a subjecthaving a thrombus or at risk of thrombus formation or to manufacture amedicament to treat a subject having a thrombus or at risk of thrombusformation.

The present invention demonstrates that a pharmaceutical composition,which comprises an effective amount of above mentioned PACAP/VIPreceptor agonist and a pharmaceutically effective carrier can be used todecrease platelet hyperfunction and/or for blocking or preventingthrombosis formation in a subject. Such pharmaceutical composition canbe used to manufacture a medicament to treat a subject having a thrombusor at risk of thrombus formation.

The invention thus provides compositions and methods useful forinhibiting, suppressing or ameliorating platelet hyperfunction inmammals, including humans. The invention has both human and veterinaryapplications. The inventive composition and method have been shown to beespecially effective in preventing thrombosis formation. A new class ofpharmaceutical compositions and methods of treatment and prevention ofthrombosis and thrombosis related injury and disease is provided.

The present invention also provides a pharmaceutical composition, whichcomprises an activator of PACAP or a PACAP receptor agonist, which incombination with an other antithrombotic agent, in an amount andproportion for enhancing the action of the other antithrombotic agent(e.g. aspirin, or an agent which blocks glycoprotein IIbIIIa, heparin,warfarin, coumarin derivatives, thrombin inhibitors, or Factor Xainhibitors) to prevent clotting or dissolve clots which have alreadyformed.

The present invention also provides a pharmaceutical composition, whichcomprises an activator of PACAP or a PACAP receptor agonist, incombination with an other antithrombotic agent, in an amount andproportion for enhancing the action of thrombolytic (e.g.staphylokinase, streptokinase, urokinase, tissue plasminogen activator,plasmin, mini- or microplasmin) to dissolve clots which have alreadyformed

In addition, the present invention finds utility in other contexts whereprevention of platelet hyperactivity is a significant concern, e.g.acute coronary syndrome, myocardial infarction, acute myocardialinfarction, unstable angina, refractory angina, peripheral vasculardisease, occlusive coronary thrombus occurring post-thrombolytic therapyor post-coronary angioplasty, a thrombotically mediated cerebrovascular,syndrome, embolic stroke, thrombotic stroke, transient ischemic attacks,venous thrombosis, deep venous thrombosis, pulmonary embolus,coagulopathy, disseminated intravascular coagulation, thrombosisfollowing angioplasty, restenosis following angioplasty, thrombosisfollowing carotid endarterectomy, thrombotic thrombocytopenic purpura,thromboangitis obliterans, thrombotic disease associated withheparin-induced thrombocytopenia, thrombotic complications associatedwith extracorporeal circulation, thrombotic complications associatedwith instrumentation such as cardiac or other intravascularcatherization, intra-aortic ballon pump, coronary stent,atherosclerosis, or cardiac valve, disorder is characterized bytransient ischemic attacks, and conditions required the fitting ofprosthetic devices.

EXAMPLES

The following examples illustrate preferred features of the invention,but are not intended to limit the invention in any way. All of thestarting materials and reagents disclosed below are known to thoseskilled in the art, and are available commercially or can be preparedusing well-known techniques.

Methodology Platelet Aggregation.

Blood was anticoagulated with 3.8% (wt/vol) trisodium citrate (9:1) andplatelet-rich plasma (PRP), obtained by centrifugation (15 minutes at150 g), was recentrifuged (3000 g for 15 min) to obtain platelet-poorplasma (PPP-trisodium citrate). Finally the platelet count in the PRPwas adjusted to 250×10⁹ platelets/L with PPP. Platelet aggregation wasperformed on two dual-channel Chrono-Log Aggregometers (ChronologCorp.), by simultaneously recording 4 tracings.

Aggregation inhibition studies were done as described before (Freson etal. (2002) Thromb. Haemost. 86:733-738; Freson et al. (2003) Hum. Mol.Genet. 15:1121-1130) and involved dose-response curves to the stableprostacyclin analogue and Gs agonist Iloprost (Ilomedine; 0-5 ng/ml;Schering), which was added to PRP 1 min prior to induction ofaggregation by Horm collagen (Nycomed, Germany; 2 mg/ml). The 50%inhibitory concentration (IC50) was calculated after curve fitting usingthe software InStat 2.03. The IC50 values±SD were compared to the IC50for the same Gs-agonist on control platelets, studied simultaneously.

Bleeding Time and Platelet Aggregation in PACAP Overexpressing Mice.

The bleeding time was determined as described before (Foster et al.(2001) J Clin Invest. 107:1591-1598.). Eight- to 15-week-old mice werebled under sodium pentobarbital anesthesia from the retro-orbitalplexus. Mouse blood was collected in a saline solution containing 20μg/mL hirudin. Preparation of PRP and aggregation were done as describedabove.

cAMP Detection in Platelets.

Platelet cAMP was measured after incubating citrated PRP with Iloprost(1 ng/ml), arresting the reaction at different time points by additionof 12% trichloroacetic acid and using a cAMP enzyme-immunoassay(Amersham, Pharmacia Biotech). Basal cAMP levels in the platelets weremeasured in the presence of the phosphodiesterase inhibitor 3-isobutyl1-methylxanthine (IBMX, 100 mM f.c.).

Human Skin Fibroblasts and cAMP Detection.

Skin fibroblasts were obtained via punch biopsy from the volar side ofthe upper arm. Fibroblasts were cultured in DMEM/H12 (Invitrogen)supplemented with 10% fetal bovine serum and antibiotics (Invitrogen),at 37° C. in a 5% CO2 humidified incubator. Only fibroblasts of lowpassage number (between 6 and 12) were used for DNA and RNA extractionand for Camp measurements. Cells were grown to 100% confluence and thenincubated in serum-free medium for 24 hours before cAMP analysis.Fibroblasts were plated in duplicate, one plate used for the assay, theother for cell counting; the obtained cAMP levels were thus adjusted forthe cell number. Patient or control fibroblasts were stimulated with theGs agonist isoproterenol (Calbiochem) at 1 mM in the presence of aphosphodiesterase inhibitor (IBMX; 100 mM) and reactions were terminatedby the cell lysis buffer supplied with the kit. The cAMP levels weremeasured using the cAMP enzyme-immunoassay mentioned above.

FISH Analysis.

FISH-analysis was performed on metaphase spreads following standardprocedures (Pinkel et al. (1986). Proc. Natl. Acad. Sci. USA. 83:2934-2938.), using a biotin labelled YAC 841C3 (Chang et al. (1993).Genomics. 17:393-402.) probe including the PACAP gene (ADCYAP1) locus at18p11.31-32 in combination with two centromeric probes for chromosome 18and 20.

Semi-Quantitative Detection of PACAP(1-38) mRNA.

Total RNA was extracted from cultured fibroblasts using TRIzol reagentaccording to the manufacturer's protocol.

Approximately 1 mg of DNasel-treated fibroblast RNA, in the presence ofan Rnasel inhibitor, was used for oligo (dT)-primed first strand cDNAsynthesis using M-MLV reverse transcriptase (RT). The reversetranscriptase reaction was terminated by heating for 5 min at 95° C. ThecDNA content was normalized using primers for b-actin. The followingprimer sets were used to generate specific fragments: b-actin beta5F5′-ACCAACTGGGACGACATGGAG-3′ [SEQ ID NO: 6] and beta3R5′-CGTGAGGATCTTCATGAGGTAGTC-3′ [SEQ ID NO: 7] and PACAP(1-38) PACAP4F5′-GAAGCACCTGCAGTCGCTCG-3′ [SEQ ID NO: 8] and PACAP 2R5′-TGTATACACAGGGTAGC-3′ [SEQ ID NO: 9]. All PCR reactions (with 17 and20 cycles) were also performed in duplicate on separate fibroblast RNAsamples.

Detection of PACAP in Plasma by ELISA.

Blood was anticoagulated with 3.8% (wt/vol) trisodium citrate (9:1) oracid citrate dextrose (ACD), pH 6.5 (9:1) and plasma (PPP) was obtainedby centrifugation for 15 minutes at 3,000 g. A polyclonal anti-PACAPantibody was coated overnight at 4° C. in microtiter plates (Costar,high binding) at 10 mg/ml in 200 ml PBS. After blocking the plates with1% nonfat dry milk in PBS, plasma samples (0-0.01 ml/200 ml) weredeposited in the wells in PBS supplemented with 1% nonfat dry milk and0.002% (v/v) Tween 80, overnight at 4° C. Bound PACAP(1-38) was revealedwith secondary horseradish peroxidase-conjugated polyclonal anti-PACAPantibody (dilution 1/2500) and O-phenylenediamine. A dilution series ofrecombinant PACAP(1-38) served as a quantitative standard. All animalexperiments were approved by the institutional review board and wereconducted according to the guidelines for animal experiments of theNational Institutes of Health.

Example 1 Generation of Megakaryocyte-Specific PACAP Overexpressing Mice

The murine GPIIb promoter (extending from +23 to 508 relative to theinitiation start site) was excised from the mGPIIb-pGL3 plasmid bydigestion with KpnI and BamHI and inserted into the KpnI-BamHI-digestedPACAP-pcDNA3.1 vector (Invitrogen) in front of the mouse PACAP gene(Denarier et al. (1993), Biochem. Biophys. Res. Commun. 195:1360-1364).The mouse PACAP gene was amplified from brain cDNA with primers mPACAP1R5′-GTAGCCGCTCGAGGATCTGCTACAAGTATGC-3′ [SEQ ID NO: 3] and mPACAP4F5′-GTTAGCCGAATTCAGTTCAAGGTCTGGCTAG-3′ [SEQ ID NO: 4], sequenced, andcloned into the EcoRI-XhoI site of the pcDNA3.1 vector. The 2.2-kilobase(kb) KpnIDraIII fragment (GPIIb-PACAP) was excised and purified forzygote injection. The GPIIb promoter has been successfully used torestrict transgene expression to the megakaryocytic cell lineage of mice(Tronick-Le Roux et al. (1995) J. Exp. Med. 181:2141-2151). TransgenicPACAP overexpressing mice were generated by zygote injection into theFriend leukemia virus, strain B (FVB) background according to previouslypublished procedures (Oury et al. (2003) Blood. 101:3969-3976; Holvoetet al. (1997) Eur. J. Biochem. 245:642-647). Transgenic offspring wereidentified by PCR screening using genomic DNA extracted from tailsamples. The following primer pair was used: mGPIIb1F5′-TGGCCACATCACAGCATTCAAG-3′ [SEQ ID No: 5] and mPACAP1R

Example 2 PACAP Overexpression in Patients A: Patient Descriptions

FIG. 1 represents a family characterised by an unbalanced segregation ofthe reciprocal translocation t(18;20) (p21;p13), of which differentmembers suffer from unexplained mental retardation. The propositus(VI:1) is a 23-year-old boy with a hypogonadotropic hypogonadism and isfollowed for epilepsy, severe mental retardation, hyperactive behaviourand hypotonia. He has an increased bleeding tendency and the Ivybleeding time was markedly prolonged (>15 minutes) but coagulationstudies are normal. Electron microscopy of his platelets is completelynormal but he presented on different occasions with a moderatethrombocytopenia, as his platelet count is always about 70-90×10³platelets/μl. His karyotype shows a partial trisomy 18p and monosomy20p. His brother (VI:2), father (V:2) and maternal grandmother (IV:6)are phenotypically normal, have no bleeding problems and have a normalkaryotype. In contrast, his mother (V:3) and maternal grandfather (IV:5)have no obvious neurologic abnormalities but a borderline IQ. They carrythe balanced translocation t(18,20) (p21,p13). They don't have anyobvious bleeding problems and have a normal platelet count. His47-year-old uncle (V:4) also suffers from severe mental retardation,pronounced recurrent epistaxis and cryptorchidism. Furthermore, hefrequently has gastric bleedings and his platelet count is around150×10³ platelets/μl. He also has a partial trisomy 18p and monosomy20p. Two other family members (IV:2 and V:1) are known with unexplainedmental retardation but from these individuals no DNA samples or furtherclinical information are available.

B: Adenylyl Cyclase Activity in Platelets and Fibroblasts

The propositus VI:1 has disturbed platelet function with a gain-of-Gsactivity measured by the platelet aggregation inhibition test, similarto patients with the XLαs insertion (Vaudry et al. (2000) cited supra;Freson et al. (2001) Thrombosis and Haemostasis 86:733-738). Plateletsfrom the patients (VI:1 and V:4) with the partial trisomy 18p/monosomy20p had a significantly increased sensitivity towards a Gs agonist, theprostacyclin analogue Iloprost (table 1), while platelets from thefamily members (IV:5 and V:3) with the balanced translocation showed amoderately increased sensitivity. The IC₅₀ value for member IV:6 withthe normal karyotype is within the rage of the IC₅₀ values from 22unrelated controls.

An important difference between patients from this family and thepatients with the XLαs insertion is their decreased sensitivity towardsthe platelet agonist such as collagen, ADP and U46619. The collagenconcentration to obtain 50% aggregation for platelets from V:4 and VI:1is significantly higher than for platelets from unrelated controls orthe normal member IV:6 (Table 1). The reactivity of platelets from IV:5and V:3 towards collagen is again mildly affected.

TABLE 1 IC₅₀ value for Iloprost in the platelet aggregation inhibitiontest IC₅₀ ± SD EC₅₀ ± SD Iloprost Collagen IV:6 0.96 ± 0.002 0.25 ±0.012 IV:5 0.50 ± 0.003* 0.74 ± 0.013 V3 0.47 ± 0.003* 0.75 ± 0.006 V40.27 ± 0.005** 1.08 ± 0.006 VI:1 0.34 ± 0.004** 1.03 ± 0.008 controls1.04 ± 0.39 (n = 22) 0.22 ± 0.6 (n = 10) IC₅₀ ± accuracy values forindicated individuals and the mean IC₅₀ value for Iloprost in theplatelet aggregation inhibition test with 2 μg/ml collagen in 22controls were calculated. A significantly (P ≦ 0.03* or P ≦ 0.0076**)lower IC₅₀ value indicates a Gs hyperfunction. The right column of thistable illustrates the significantly decreased response to collagen(μg/ml) for respectively IV:5 and V:3 (P ≦ 0.003*) versus V:4 and VI:1(P ≦ 0.0001**) compared to 10 controls or IV:6. EC₅₀ is expressed ascollagen concentration that induces aggregation with amplitude 50% ofmaximal aggregation.

For patients with the XLαs insertion, it has been shown that thefunctional responses mediated by stimulation of Gs agonists are due tohyperactivity of adenylyl cyclase only when Gs-coupled receptors arestimulated (Vaudry et al. (2000) cited supra; Freson et al. (2001) citedsupra). These patients had normal basal cAMP levels. This indicates thatin the platelets of the propositus VI:1, adenylyl cyclase is alreadyactivated under basal conditions. In addition to an increased cAMPresponse to Iloprost, patient VI:1 indeed shows higher basal cAMP levels(FIG. 2A). cAMP levels were measured in fibroblasts from VI:1. A similarincreased basal and stimulated cAMP response (FIG. 2B) was found.

C: PACAP(1-38) mRNA and Protein Overexpression

Patient VI:1 has a normal Gsα mRNA and protein expression level and thecoding sequence for the Gsα gene and XL-exon1 were completely normal.

Since this patient had a partial trisomy 18p and monosomy 20p, thesechromosomes were screened for candidate genes. Interestingly,measurement of the adenylyl cyclase activity in fibroblasts from anunrelated patient with a complete trisomy 18 showed similarly increasedbasal and stimulated cAMP levels (FIG. 2B). The gene for PACAP (ADCYAP1)is located on chromosome 18p31-32 (Hosoya M. et al. Biochim Biophys Acta(1992) 1129, 199-206.) and is a possible candidate since its activepeptide, PACAP(1-38), stimulates Gs-coupled receptors and therebyactivates adenylyl cyclase. FISH analysis with YAC clone Y841C3 (ChangE. et al. Genomics (1993) 17: 393-402.), that contains ACDYAP1, showedthat the translocation results in three copies of the gene in patientsVI.1 and V:4.

Human skin fibroblasts express PACAP(1-38) and the PACAP type1-receptor(VPAC1) (Steinhoff M, et al. Regul Pept (1999), 80: 49-55). PACAP(1-38)mRNA was overexpressed in fibroblasts from patient VI:1 (FIG. 3A) bysemi-quantitative RT-PCR. No PACAP mRNA was found in platelets byRT-PCR, probably due to their unstable RNA. However, western blotanalysis showed that platelets express the VPAC1 receptor. The activepeptide PACAP (1-38) is mainly expressed in testis and brain but thispeptide can cross the blood-brain barrier and is stably transported inplasma through coupling with ceruloplasmin (Banks W A, J Pharmacol ExpTher (1993) 267: 690-696; Tams JW, Biochem J (1999) 341: 271-2768, 9).PACAP is expressed in the human megakaryocytic cell lines MEG-01, DAM1and K562 and a rather weak expression was found in control platelets byRT-PCR (FIG. 3B). Western blot analysis further revealed that plateletsexpress the VPAC1 receptor, the levels of which were normal in thepatient (FIG. 3C). PACAP(1-38) was detected in human plasma by ELISA andsignificantly higher levels were found in patients VI:1 and V:4, andmoderately increased levels in IV:5 and V:3, in contrast to a plasmapool of unrelated controls or IV:6 (FIG. 3D). Platelet aggregation usingwashed control platelets resuspended in citrated plasma from a controlor patient VI:1, indicated that plasma from VI:1 inhibits the collageninduced aggregation (FIG. 3D). This could be due to the increased amountof PACAP(1-38) in this plasma.

D: Role for PACAP in Platelet Aggregation

To establish the role of circulating PACAP(1-38) as a physiologicalinhibitor of collagen-induced platelet aggregation, various approacheswere taken using different models. We made transgenic mice with a PACAPoverexpression in the megakaryocyte lineage. As for the patients, thesemice presented with an increased bleeding time and showed a decreasedsensitivity towards collagen-induced aggregation (FIG. 4A). In anotherset of experiments, the patient phenotype was reversed, i.e. theactivity of PACAP was neutralized by addition of the PACAP antagonistPACAP(6-38). This recombinant peptide has a 10-100 times higher affinityfor the VPAC1 receptor than PACAP(1-38) but seems not to activateadenylyl cyclase (Vaudry et al., (2000) cited supra). PACAP(6-38)enhanced the collagen-induced aggregation of normal human platelets in adose-dependent manner (FIG. 4B) and decreased basal cAMP levels (datanot shown). The PACAP antagonist also induced a partial but consistentimprovement of the collagen-induced platelet aggregation in the patient(FIG. 4B).

The functional platelet aggregation inhibition test for a control personin the presence of PACAP (6-38) results in a Gs loss-of-function (FIG.4B). The influence of PACAP(6-38) on the platelet aggregation test forpatient VI:1 was not that pronounced, probably because his PACAP plasmalevels were too high.

Example 3 Influence of PACAP in Platelet Aggregation by Studies in Mice

Functional platelet studies from patient VI:1 show that increased levelsof PACAP(1-38) in plasma result in increased basal cAMP levels and aplatelet hypofunction. The role of PACAP(1-38) in platelet function wasalso studied in mice by subcutaneous injection of polyclonal ormonoclonal anti-PACAP antibodies.

FVB mice were injected three times subcutaneously with 200 mg of ananti-PACAP or a control anti-b2-glycoprotein I polyclonal antibody orfour times with 50 mg of an anti-PACAP (PP1A4) or an anti-RGS2monoclonal antibody with an interval of 3 days between injections. Atday 14 after the first injection, whole blood from the inferior venacava of mice anesthetized by intraperitoneal injection of 60 mg/kgsodium pentobarbital was drawn into 20 μg/mL hirudin. A platelet countwas performed on the venous blood sample using the Cell Dyne 1300(Abbott). Blood was centrifuged at 100 g for 10 min, allowing separationof PRP, and PPP was obtained by centrifugation of the remaining blood at2,000 g for 10 min. PRP and PPP were pooled from 5 mice in each group.Platelet aggregation was measured as described above with the plateletcount adjusted to 250×10⁹ platelets/L.

The antibodies had a similar effect on platelet aggregation as the PACAPantagonist PACAP(6-38). Platelets incubated with anti-PACAP antibodies(10 μg/ml) show an enhanced response towards collagen stimulation (FIG.5). Moreover, when mice were functionally tested by platelet aggregation7 days after their last injection, mice treated with anti-PACAPantibodies show the opposite phenotype to that observed in patient VI:1.In contrast to the treatment with the aspecific antibody againstβ2-glycoprotein I, anti-PACAP-treated mice show a weaker responsetowards activation of the Gs pathway and have an enhanced responsetowards collagen stimulation (FIG. 5A, B).

Example 4 Influence of PACAP in Thrombocytopenia

Functional platelet studies on patient VI:1 show that increased levelsof PACAP(1-38) result in increased basal platelet cAMP levels andplatelet hypofunction. Since this patient also was thrombocytopenic, wehave investigated whether elevated PACAP(1-38) would be associated withdefective platelet formation. Platelets from this patient are smaller asthe mean platelet volume (MPV) was 8.2 fL (normal MPV: 9-13 fL) and FACSanalysis of his platelets showed a reduced expression of glycoprotein IX(FIG. 5D). Morphological examination of the bone marrow from the patientrevealed a moderate to normal presence of the megakaryocyte lineage,indicative also of a normal proliferation. Electron microscopy of 2.5%glutaraldehyde fixed and routinely prepared ultrathin sections showedthe presence of megakaryocyte progenitors but no mature megakaryocytes.The immature megakaryoblasts seemed to have reduced levels of roughendoplasmic reticulum cisternae and free ribosomes (FIG. 5E). Cleardemarcation membranes were not seen.

Specific granules as well as some dense core vesicles were obvious. Bonemarrow from PACAP overexpressing and control mice was grown for 11 daysin the presence of TPO, IL-6, IL-1b and SCF (Blair et al. (20022) Br. J.Haematol. 116:912-919.); FACS analysis showed a reduced expression ofglycoprotein IIIa in megakaryocytes from the transgenic animals (FIG.5D), indicative of a maturation defect. Transgenic mice present with anormal platelet count but their platelets have reduced levels ofglycoprotein IIb/IIIa.

The role of PACAP(1-38) in thrombopoiesis was therefore further studiedin control mice by subcutaneous injection of neutralizing polyclonal ormonoclonal anti-PACAP antibodies. Mouse platelet-rich-plasma incubatedwith anti-PACAP antibodies in vitro (10 mg/ml) show an enhanced responsetowards collagen (data not shown), similar to the findings with thePACAP antagonist PACAP(6-38) using human platelets (FIG. 4B). Moreover,7 days after the last injection, anti-PACAP antibody treated mice showan enhanced response towards collagen ex vivo (FIG. 5B). Mice injectedwith the anti-PACAP polyclonal antibodies (n=5) furthermore haveincreased platelet numbers incomparison to the control group (n=5)(1194±237×109 plt/L versus 722±178×109 plt/L, p=0.01-unpaired T-test) 14days after the first injection. This experiment was repeated determiningplatelet numbers at different time points (days 0, 3, 7, 9, and 14), viatail bleeding (FIG. 5F). Mice injected with anti-PACAP polyclonalantibodies already showed increased platelet numbers 3 days afterantibody injection.

The increased thrombocytopoiesis after pre-treatment with a polyclonalanti-PACAP antibody was studied under conditions of chemicallysuppressed bone marrow by the agent busulfan. This was done bysubcutaneous injection of mice with either a polyclonal anti-PACAP or acontrol polyclonal antibody (at days 0, 3, and 7) and afterwards anintraperitonal injection of Busulfan (20 mg/kg) (at days 8 and 11). Theplatelet number was counted at different time points. Mice pretreatedwith the polyclonal anti-PACAP antibody recovered more rapidly fromtheir thrombocytopenic condition than the mice injected with the controlantibody (FIG. 8A, B).

Example 5 Generation of Polyclonal and Monoclonal Antibodies

The generation of monoclonal antibodies is exemplified in extenso forPACAP (1-38). The same methodology can be used for shorter versions ofPACAP (n terminally or c terminally truncated). The same methodology canalso be used for a full length PACAP receptor or for a fragment thereof.Preferred fragments of a PACAP receptor are the extracellular domains ofthese receptors.

A recombinant human PACAP(1-38) fusion protein, consisting of the aminoacids encoded by the PACAP(1-38) peptide coupled to GlutathioneS-transferase (GST) was expressed in Escherichia coli and purified byaffinity chromatography on immobilized glutathione (AmershamBiosciences). Recombinant human PACAP(1-38) is mixed with an equalamount of an adjuvant, and an obtained mixture is than subcutaneouslyadministrated to Balb/c male mice (8 weeks old upon the start ofimmunization) in an amount corresponding to an amount of PACAP(1-38) of100 μg per 1 mouse (priming immunization). After about 21 days,immunization can be performed by subcutaneous administration in the samemanner as described above (booster immunization). After 19 days or 30days from the booster, the mice can administrated through their tailveins with 200 μl of a preparation obtained by diluting humanPACAP(1-38) with PBS (phosphate-buffered physiological saline) to have aconcentration of 250 μg/ml (final immunization). Spleens have than to beexcised from the mice after about 3 days from the final immunization,and they have to be separated into single cells. Subsequently, thespleen cells should be washed with a proper medium, e.g. DMEM medium. Onthe other hand, suitable mouse myeloma cells (e.g. Sp2/0-Ag14) have tobe collected in the logarithmic growth phase, and to be washed with aproper medium, e.g. DMEM medium. The spleen cells and the mouse myelomacells have to be sufficiently mixed in a plastic tube in a ratio ofnumbers of the cells of 10:1, followed by addition of 50% (w/v)polyethylene glycol (PEG e.g. of Boehringer Mannheim, average molecularweight: 4000) to perform cell fusion at 37° C. for 7 minutes. Afterremoval of the supernatant solution (by means of centrifugation), theresidue is added with HAT medium (DMEM medium containing 10% fetalbovine serum added with hypoxanthine, aminopterin, and thymidine). Theresidue has to be suspended so that a concentration of the spleen cellsof about 5×106 cells/ml is obtained. This cell suspension can than bedispensed and poured into 96-well plastic plates so that one wellcontains about 100 μl of the suspension, followed by cultivation at 37°C. in 5% carbon dioxide. HAT medium has to be supplemented; for instancein an amount of 50 μl/well on 2nd and 5th days. After that, half volumeof the medium can be exchanged every 3 or 4 days in conformity withproliferation of hybridomas.

Screening and Cloning of Hybridomas: Hybridomas, which produce themonoclonal antibody of the present invention, have to be screened for.This has to be done by using, as an index, the inhibitory activity ofthe monoclonal antibody on the physiological activity possessed byPACAP. Hybridomas, which produced monoclonal antibodies exhibitingreactivity with PACAP's have then to be selected from the selectedclones. The obtained hybridomas have then to be transferred to asuitable medium for instance HT medium which is the same as HAT mediumexcept that aminopterin is removed from HAT medium, and culturedfurther. Cloning can be performed twice in accordance with the limitingdilution method by which stable hybridomas are obtainable.

Production and Purification of Monoclonal Antibodies:2.6,10,14-Tetramethylpentadecane (e.g. Pristane of Sigma, 0.5 ml) can beintraperitoneally injected into Balb/c female mice (6 to 8 weeks oldfrom the birth). After 10 to 20 days, cells of clones can be (1×10⁶ to10⁷ cells) suspended in PBS and intraperitoneally inoculated into themice. After 7 to 10 days, the mice can be sacrificed and subjected to anabdominal operation, from which produced ascitic fluid can be collected.The ascitic fluid can be centrifuged to remove insoluble matters, and asupernatant was recovered and stored at −20° C. until purificationConsequently, IgG can be purified from the ascitic fluid supernatantdescribed above by using Hi-Trap Protein-A antibody purification kit(available from Pharmacia, Roosendaal, Netherlands). Namely, the asciticfluid (2 ml) can be added with Solution A (1.5 M glycine, 3 M NaCl, pH8.9, 8 ml), and filtrated with a filter for filtration having a poresize of 45 μm (Millipore). After that, an obtained filtrate can appliedto a column (column volume: 1 ml) charged with Protein Sepharose HP(produced by Pharmacia) sufficiently equilibrated with Solution A, andthe column has be washed with Solution A in an amount of 10-fold columnvolume. Subsequently, an IgG fraction can be eluted with Solution B (0.1M glycine, pH 2.8) in an amount of 10-fold column volume. The eluted IgGfraction can be dialyzed against PBS. The monoclonal antibodies can bedetermined for their IgG subclasses by using the purified antibodiesobtained in the foregoing, by means of a commercially availablesubclass-determining kit (trade name: Mono Ab-ID EIA Kit A, produced byZymed). This method is based on the ELISA method. The InhibitoryActivities of Monoclonal Antibodies can be tested for their possiblestimulatory effect on collagen-induced (0.35 μg/ml) platelet aggregationof human or mouse platelets. A similar approach may be used for thepreparation of monoclonal antibodies specific to VIP or to PACAPreceptor VPAC1 or VPAC2 or fragments thereof. Such antibodies can beapplied for dosing activating or inhibitory PACAP mimetics by ELISA, forthe purpose of monitoring PACAP (analogues) concentrations in the plasmaof treated subjects. The VPAC1 have been cloned (Harmar et al. (1998)Pharmacol Rev. 50: 265-270). The human VPAC1 receptor DNA has beencharacterised from a HT29 human colonic adenocarcinoma cell linelibrary. Human VPAC1 receptor comprises 457 amino acids (Sreedharan etal. (1993) Proc. Natl. Acad. Sci. USA 92: 2939-2943). The human VPAC1receptor gene is located on region p22 of chromosome 3 (Sreedharan etal. (1995) Biochem Biophys Res Commun 193: 546-553). Cell lines, suchSaccharomyces cerevisae, which are naturally devoid of VPAC1, can betransfected to produce such (Hansen MK (1999) Receptors Channels 6:271-281). Vectors for expression of a PACAP receptor have been describedin WO0107478. The method for preparation of a PACAP receptor protein hasbeen disclosed in patent application US20020155533.

Preparation of F(ab′)2 or monovalent Fab fragments: In order to prepareF(ab′)2 fragments, the monoclonal antibody can be dialyzed overnightagainst a 0.1 mol/L citrate buffer (pH 3.5). The antibody (200 parts)are then digested by incubation with pepsin (1 part) available fromSigma (Saint-Louis, Mo.) for 1 hour at 37° C. Digestion is consequentlystopped by adding 1 volume of a 1 M Tris HCl buffer (pH 9) to 10 volumesof antibody. Monovalent Fab fragments can prepared by papain digestionas follows: a 1 volume of a 1M phosphate buffer (pH 7.3) is added to 10volumes of the monoclonal antibody, then 1 volume papain (Sigma) isadded to 25 volumes of the phosphate buffer containing monoclonalantibody, 10 mmol/l L-Cysteine HCl (Sigma) and 15 mmol/L ethylenediaminetetra-acetic acid (hereinafter referred to as EDTA). Afterincubation for 3 hours at 37″ C, digestion is stopped by adding a finalconcentration of 30 mmol/l freshly prepared iodoacetamide solution(Sigma), keeping the mixture in the dark at room temperature for 30minutes. Both F(ab′)2 and Fab fragments can further be purified fromcontaminating intact IgG and Fc fragments using protein-A-Sepharose. Thepurified fragments can finally dialyzed against phosphate-bufferedsaline (herein after referred as PBS). Purity of the fragments can bedetermined by sodiumdodecylsulphate polyacrylamide gel electrophoresisand the protein concentration can be measured using the bicinchonicicacid Protein Assay Reagent A (Pierce, Rockford, Ill.).

1. A method of treating thrombocytopenia in a subject havingthrombocytopenia, said method comprising the step of administering tosaid subject an inhibitor of the PACAP signalling via the VPAC1receptor.
 2. The method according to claim 1, wherein saidthrombocytopenia is infection-induced thrombocytopenia ortreatment-induced thrombocytopenia.
 3. A method of preventingthrombocytopenia in a subject having a high probability of acquiring ordeveloping thrombocytopenia, said method comprising the step ofadministering to said subject an inhibitor of the PACAP signalling viathe VPAC1 receptor.
 4. The method according to claim 3, wherein saidsubject has cancer and wherein said compound is administered prior orduring chemotherapy.
 5. The method according to claim 3, wherein saidsubject is at risk of acquiring or developing infection-inducedthrombocytopenia by exposure to infectious agents.